Summary

In the Drosophila leg, activation of Notch leads to the
establishment of the joints that subdivide the appendage into segments. We
find that mutations in bowl result in similar phenotypes to Notch,
causing fusion and truncations of tarsal segments (tarsomeres) and, like its
close relative Odd-skipped, Bowl is produced in response to Notch signalling
at a subset of segment boundaries. However, despite the fact that
bowl mutant clones result in fusion of tarsomeres, Bowl protein is
only found at the t1/tibial and t5/pretarsal boundaries, not at tarsomere
joints. One hypothesis to reconcile these data is that bowl has a
role at an earlier stage in tarsal development. We therefore investigated the
effects of bowl mutations on the expression of leg `gap' genes that
confer regional identity on the developing leg. Several of these genes have
altered expression in bowl mutant cells. For example,
bric-a-brac2 is normally expressed in the central part of the tarsus
domain but expands into distal and proximal regions in bowl clones.
Conversely, ectopic bowl leads to a reduction in
bric-a-brac2, with a concomitant expansion of proximal (t1) and
distal (t5) tarsomere fates. The bowl gene is therefore required for
the elaboration of pattern in the tarsus and its effects suggest a progressive
model for the determination of P/D identities. This mechanism might be
important in the diversification of arthropod limbs, because it explains how
segmented tarsomeres could have arisen from an ancestral limb with an
unsegmented tarsus.

Introduction

Animal limbs develop as outgrowths from the main body axis that acquire
proximal/distal (P/D) patterning to form a series of specialized skeletal
structures. These structures are articulated and so one key consequence of P/D
patterning is the establishment of joints between each skeletal element. In
the Drosophila leg, the P/D axis is established through the combined
activities of Wingless (Wg) and Decapentaplegic (Dpp), which intersect in the
centre of the limb primordium. Wg and Dpp together induce the expression of
Distal-less (Dll), a homeodomain protein required for the development of all
distal leg structures (Cohen et al.,
1989; Diaz-Benjumea et al.,
1994), and Dachshund (Dac), a nuclear protein required for
intermediate leg segments (femur and tibia)
(Mardon et al., 1994;
Lecuit and Cohen, 1997). By
the beginning of the third larval instar, the leg primordium is therefore
subdivided into at least three regions. Subsequent patterning involves
interactions between the factors expressed in these early territories. For
example, several genes are required for the development of the tarsus,
including rotund and bric-a-brac
(Kerridge and Thomas-Cavallin,
1988; Agnel et al.,
1989; Godt et al.,
1993; Chu et al.,
2002; Couderc et al.,
2002; Galindo et al.,
2002; St Pierre et al.,
2002). Expression of these genes is promoted by Dll and restricted
by the combined activities of Dac proximally and a gradient of
epidermal-growth-factor-receptor signalling distally
(Campbell and Tomlinson, 1998;
Campbell, 2002;
Galindo et al., 2002). By the
stage that a series of P/D regions have been established, further patterning
appears to be independent of the initial inducers Wg and Dpp
(Lecuit and Cohen, 1997;
Galindo et al., 2002).
However, it is not clear how these P/D regions are elaborated (for example, to
give diversity to the distal tarsal structures).

A final stage in translating the P/D patterning into the definitive
segmented structure of the insect adult leg is the formation of the
inter-segmental joints. The leg consists of six true segments or podites
(coxa, trochanter, femur, tibia, tarsus and pretarsus), which are
independently moveable by muscles. In Drosophila, the tarsus is
further subdivided into five tarsomeres (t1-t5), which have distinct
characteristics but lack independent musculature
(Snodgrass, 1935). Development
of both `true' joints and inter-tarsomere joints requires Notch activity,
shown by the loss of joints and fused segments in Notch mutant cells,
and by the ectopic joints that are formed when extra sites of Notch activity
are engineered (de Celis et al.,
1998; Bishop et al.,
1999; Rauskolb and Irvine,
1999). Consistent with its pivotal role in specifying joint
development, Notch activity is detected at all segment/subsegment boundaries
at the end of larval development, using transcription of the Enhancer of
split target genes as a measure (de
Celis et al., 1998). However, expression of Notch ligands is first
observed at a subset of locations at a much earlier stage shortly after the
initial `regional' domains of gene expression are established
(Rauskolb, 2001). There are
two explanations for this. One is that the specification of joints occurs
sequentially, with some joints being determined early and others (e.g.
tarsomere joints) much later. Alternatively, Notch activity might have both
earlier roles in P/D regionalization and patterning and later roles that build
on these earlier events to establish the segmental boundaries and joints at
the correct locations.

To investigate further the mechanisms involved in P/D limb development, we
have looked for genes whose expression is dependent on Notch activity that
could allow us to establish whether it has roles in the initial P/D patterning
as well as in the subsequent establishment of joints. The zinc-finger protein
encoded by the gene bowl is detected at a subset of sites of Notch
activity and its expression is dependent on Notch. The bowl gene is
closely related to the segmentation gene odd-skipped, and is required
for development of the embryonic hindgut
(Wang and Coulter, 1996;
Iwaki et al., 2001). Our
analysis of bowl and odd-skipped function in the developing
leg indicates that these genes are involved in the elaboration of pattern in
the tarsus, leading us to propose that Notch is important for patterning as
well as for joint formation. The effects of Bowl on tarsal development suggest
that P/D tarsal identities are determined progressively and might also explain
how different numbers of tarsomeres could have arisen from an ancestral limb
that is thought to have contained an unsegmented tarsus
(Snodgrass, 1935).

Materials and methods

Genetics

Except where otherwise stated, fly stocks used are described in FlyBase.
For analysis of Bowl, we used both bowl2, a strong
loss-of-function allele (H284Y) (Wang and
Coulter, 1996) and bowl1, a null allele
(S232@) (Wang and Coulter,
1996). We also analysed three P elements inserted within 100 bp of
the transcription start site, including bowlk08617. None
correspond to the bowl alleles because they fully complement
bowl2 and Df(2R)ed1. The
lacZ/Gal4 pattern of expression in imaginal discs also differs
between each P element, so we have not used these further. The lacZ
insertion in odd is P{lacZ}oddrK111 and
odd5 is a strong hypomorph.
E(spl)mβ-CD2 and
E(spl)mβ1.5-lacZ are reporter genes that mimic
E(spl)mβ transcription (de
Celis et al., 1998).

The bowl or odd alleles were recombined onto
w1118; P{mW+mw=pM}36F
P{ry+t7.2=neoFRT}40A and were crossed to the following marked
strains for inducing clones:

f36a hsFLP; ck {f+}
P{ry+t7.2=neoFRT}40A/CyO

f36a hsFLP; P{ry+ y+}25F
P{ry+t7.2=neoFRT}40A/CyO

f36a hsFLP; P{ubi-GFP} P{ry+t7.2=neoFRT}40A

Clones were induced by a 1-hour heat shock at 38°C at 48-72 hours of
development. Large mutant clones were also induced by X-irradiation at a dose
rate of 1.28 Rads per second for 900 seconds using the Minute stock
f36a; M(2)z P{f+}30B/CyO.

UAS-bowl was generated using the full-length cDNA clone LD15614
obtained from the Berkeley Drosophila Genome Project. The
bowl cDNA was excised using NotI and XhoI, and
ligated into pUAST digested with the same enzymes. Transgenic flies were
obtained by injection into y w, following standard P-element
transformation procedures. Independent lines were analysed and graded
according to the strength of phenotypes elicited with GAL4 drivers as follows:
UAS-bowl1.1 (strong) > UAS-bowl6.1
(moderate) > UAS-bowl9.1 (weak)

Results

Bowl and Notch produce similar segmentation and growth defects in the
leg

In the developing leg, Notch mutant cells result in fused leg
segments, owing to the absence of joints, and in severely reduced growth
(Fig. 1A-C)
(Shellenbarger and Mohler,
1978; de Celis et al.,
1998; Bishop et al.,
1999; Rauskolb and Irvine,
1999). Accumulation of pigmented tissue also occurs at joints in
proximal regions (data not shown). Mutations in bowl result in
similar phenotypes; the mutant cells are associated with fusions and
truncations of tarsal segments as well as with melanotic patches at the
proximal joints (Fig. 1D-F;
Fig. 2). Because the gene is
essential at earlier stages in development (bowl mutant embryos do
not hatch; Wang and Coulter,
1996), it is difficult to determine the consequence of completely
eliminating bowl in the leg. However, when the mutant clones cover
most of the distal part of the leg, the limb is severely truncated, with
little or no segmentation/joint tissue evident
(Fig. 1E and data not shown).
Conversely, clones that are restricted to the central part of the tarsus often
fail to result in a detectable phenotype
(Fig. 2).

Phenotypes are only detected in bowl clones spanning several
tarsomeres. Analysis of 62 legs with bowl2 clones marked
by absence of y. Diagrams depict the segments affected in each clone
(tibia and tarsomeres t1-t5). 34% of legs analysed had aberrant tarsomeres
(green shading indicates extent of clone; specific tarsomeres showing defects
are listed, with boxed text indicating segment fusions). 66% of legs
containing clones appeared normal (yellow shading indicates extent of clone).
Only clones that include t1 (or t5, data not shown) give rise to tarsomere
fusions; phenotypes are not detected in clones that only span tarsomere
boundaries.

The bowl gene encodes a zinc-finger transcription factor and is
closely related to odd-skipped, a gene that has already been
implicated in leg segmentation (Wang and
Coulter, 1996; Rauskolb and
Irvine, 1999). We therefore examined phenotypes produced by
odd mutant cells and found similar defects to bowl –
segmental fusions and truncations in the tarsal region, and melanotic patches
in proximal joints (Fig. 1G,H).
The two genes therefore have similar but essential roles in leg segmentation,
although their functions elsewhere appear to be distinct
(Wang and Coulter, 1996).

Given the profound effect on tarsal segmentation and similarities with
Notch phenotypes, we expected that bowl would be expressed
at the sites where Notch is active in the tarsus. We therefore compared the
expression of bowl with E(spl)mβ, a known target of
Notch signalling in the leg, using an E(spl)mβ-lacZ
transgene (Cooper et al., 2000)
and an antibody that recognizes Bowl. Although Bowl and β-galactosidase
are clearly co-expressed at some positions, including the t5/pretarsus
boundary and the tibia/t1 boundary, Bowl was not detected at sites of Notch
activity within the tarsus (Fig.
1I-I″). Indeed, the distribution of Bowl and Odd appears to
be identical and neither is detected at tarsomere boundaries
(Fig. 1J-K″)
(Rauskolb and Irvine, 1999).
Both are present at all the proximal joints (coxa/femur, femur/tibia,
tibia/t1) and at a distal site, the t5/pretarsal boundary
(Fig. 1J,K; the latter has not
previously been documented as a site of Notch activity, although it clearly
expresses E(spl)mβ and gives rise to an articulated joint). In
summary, therefore, Bowl and Odd are present at a subset of the segmental
boundaries where Notch is active in the developing leg. These correspond to
the boundaries between `true' segments and not to those between
tarsomeres.

bowl is regulated by Notch in the developing leg

Expression of the Notch ligands is a key step in regulating Notch activity
in the developing leg (Mishra et al.,
2001; Rauskolb,
2001). To investigate the relationship between Bowl and Notch
activity, we first compared the timing and distribution of Bowl expression
with that of Serrate and Delta, which both regulate Notch activity in the leg
disc (Mishra et al., 2001;
Rauskolb, 2001). By monitoring
expression from early third instar, we found that the evolution of
Bowl/odd-lacZ expression closely parallels that of the Notch ligands
(Fig. 3A-D). The only
significant discrepancy appears late in the third instar, when Serrate and
Delta are detected at intertarsomere boundaries but Bowl and odd-lacZ
are not (Fig. 3C,C′ and
data not shown). Before that stage, Bowl/Odd expression occurs distal to each
domain of Delta that is established. For example, the central t5/pretarsal
ring of Bowl appears at ∼86 hours (Fig.
3A-C) and correlates with the appearance of Delta in the tarsus
and a transient expression of Serrate on the distal, pretarsal, side
(Fig. 3A-C and data not shown)
(Rauskolb, 2001).

We then tested more directly whether Bowl accumulation at segment
boundaries depends on Notch activity, by generating clones of Notch
mutant cells in the disc epithelium. In all cases in which Notch clones
crossed between t5 and the pretarsus, the ring of nuclear Bowl protein at the
boundary was interrupted (15/15; Fig.
3E). The effects at the t1/tibia and tibia/femur boundaries were
less clear cut, with some clones showing absence (6/21) or reductions (7/21)
in Bowl, whereas others retained apparently wild-type levels (8/21). Many of
the last group were small clones (seven cells or less; 5/8). In converse
experiments, expression of a constitutively activated form of Notch
(Notchicd) resulted in ectopic Bowl accumulation at a subset of
locations in the disc (Fig.
3F). These broadly correspond to the areas where Bowl is normally
detected. Taken together, these data indicate that Bowl is responsive to Notch
regulation but that the regulation is limited to a specific time window and/or
position. Similar results have been obtained with odd, which is only
responsive to Notch in selected regions
(Rauskolb and Irvine,
1999).

Mutations in bowl alter the expression of genes involved in
tarsal patterning

Neither Bowl nor Odd appear to be present within the tarsus
(Fig. 1J-K,
Fig. 3C)
(Rauskolb and Irvine, 1999),
yet mutations in either gene produce defects in this part of the leg (fusion
of tarsomeres and growth defects; Fig.
1D-H). There are three models to explain this. (1) Bowl and Odd
influence tarsomere segmentation indirectly, by regulating production of a
long-range signal. (2) The two genes are expressed at intertarsomere joints
but at a level too low to be detected. (3) They are involved in an earlier
patterning event that influences subsequent tarsal segmentation. We can rule
out the first hypothesis, because fusions only occur within tarsal
bowl clones (Fig.
1D,F; Fig. 2) and
the effects on downstream genes are autonomous, as is clearly seen in
Fig. 4 (e.g.
Fig. 4D). Although it is
difficult to rule out the low levels of tarsal expression implied by the
second model, our data argue that this is an unlikely explanation for several
reasons. First, bowl clones that only spanned intertarsomere joints
(e.g. t2/t3/t4) appeared normal (Fig.
2). Almost all clones that resulted in observable phenotypes
spanned the tibia/t1 boundary and, even within these clones, there was
considerable variation in the number of tarsomere joints affected
(Fig. 2). This suggests that
the effect on tarsomere joints is a secondary consequence of the mutations.
Second, in the case of Odd, the pattern of expression detected in the larva
persists throughout pupal leg morphogenesis
(Mirth and Akam, 2002), ruling
out later expression in the tarsomeres. Third, when we produce high levels of
bowl mRNA using GAL4/UAS system, very little protein is detected in
the tarsal domain of late-third-instar/early-pupal legs, suggesting either
that the protein is unstable or that the mRNA is poorly translated in this
region (e.g. see Fig. 6 and
data not shown).

Ectopic bowl expression causes expansion of proximal and distal
tarsal fates. (A-D) Expression of bowl within distal leg leads to
fusion of segments and transformation to more proximal t1 fates as indicated
by ectopic sex combs (e.g. arrowheads). (A) Wild-type prothoracic leg; arrow
marks the sex comb in t1. (B-D) Prothoracic legs from lines with different
levels of bowl expression: (B) weak,
klumpfuss-Gal4G410/UAS-bowl[6.1]; (C)
intermediate, Dll-Gal4em212/UAS-bowl[9.1]; (D)
strong, Dll-Gal4em212/UAS-bowl[1.1]. Higher
levels of Bowl lead to more severe phenotypes: in (D), the tarsus is
completely fused with multiple ectopic sex combs. Domain of Dll
expression is seen in Fig.
4A-C; klumpfuss-Gal4G410 is in patches and
rings within the tarsal region (Klein and
Campos-Ortega, 1997). (E,F) Expression of Dac (red), Bab2 (blue)
and BarH1 (green) in pupal legs from wild-type (E) and
Dll-Gal4em212/UAS-bowl[9.1] (F). Ectopic Bowl
results in decreased Bab2 and expansion of Dac (red arrowhead) and BarH1
(green arrowhead). (G-G″) Levels of ectopic Bowl protein (green) are
more variable than those of β-galactosidase (red), even though both are
driven by ptc-Gal4. (120 hour leg discs from
Ptc-Gal4559.1/UAS-bowl[1.1], UAS-lacZ). Only low levels of
Bowl are detected within the tarsus or pretarsus (arrowheads). Expression of
bowl mRNA is more uniform and similar to lacZ (data not
shown). (H,H′) Prothoracic leg from
ssa/ss114 has ectopic sex comb on t2 (H,
arrowhead), ectopic joint in t1 (H′, arrow) and fusion of t2-t3
(H′, arrowhead). (I,I′) In
ssa/ss114 pupal leg discs, ectopic patches of
Bowl (green) are seen in t1 and t2 (arrowheads). Dac (red) is unaffected but
Bab (blue) is decreased in places (*).

To investigate the third model (that bowl has an early patterning
function in the leg), we asked two questions. First, we tested whether
E(spl)mβ-CD2 expression in t2-t4 is affected by
bowl mutations, as predicted if Bowl acts prior to tarsomere boundary
formation. As in the adult legs, there is considerable variation between
clones, but we observed clear disruptions to
E(spl)mβ-CD2 in three out of nine clones
(Fig. 4B-B″). These
defects are confined to the clone but do not strictly follow clone boundaries
because some of the mutant cells retain wild-type levels of
E(spl)mβ-CD2 even in the most severe cases
(Fig. 4B″, insert). This
demonstrates that bowl function precedes Notch activation at the
tarsomere boundaries and supports the hypothesis that its effects on tarsomere
boundaries are secondary.

Second, we asked whether mutations in bowl alter the expression of
genes involved in the initial regionalization of the leg. Several genes have
been identified that confer distinct regional identities and are expressed in
broad domains within the leg disc. These include dachshund
(dac), which is expressed in more proximal regions including t1
(Mardon et al., 1994;
Lecuit and Cohen, 1997), the
two genes of the bric-a-brac (bab) complex (bab1
and bab2), which are expressed in the presumptive tarsal region
(Godt et al., 1993;
Couderc et al., 2002;
Galindo et al., 2002), and two
Bar genes that are expressed in distal tarsal segments t4 and t5
(BarH1 and BarH2)
(Kojima et al., 2000). We find
that expression of all three types of regional genes is affected by mutations
in bowl. In late-third-instar/early-pupal leg discs, Bab2 expression
normally extends to the proximal edge of t1
(Fig. 4A). In bowl
clones, ectopic Bab2 is detected in proximal parts of t1 and the levels in t2
are also altered (Fig. 4B,D,E).
Conversely, when these clones also encompass the distal part of Dac domain,
there is a reduction in Dac (Fig.
4C,D). Mutant clones that lie at the distal side of the tarsal
domain again show derepression of Bab2 (in distal t5, where Bab2 expression is
low or absent), and this is coupled with a reduction in the levels of BarH1
(Fig. 4F). In all cases, the
effects are autonomous to the clone and precisely follow clone boundaries.

We therefore conclude that Bowl regulates the expression of patterning
genes, promoting development of the proximal (t1/t2)
(Fig. 4B,D,E) and distal (t5)
extremities of the tarsus (Fig.
4F). Thus, bowl mutations lead to an expansion of a
`central tarsal fate' that is characterized by uniform Bab2 and decreased
BarH1 and Dac. This disruption in tarsal patterning would in turn affect the
expression of Notch ligands in the tarsus and hence lead to defects in
tarsomere joints, as seen in the disruption of
E(spl)mβ-CD2 (Fig.
4B). However, there is still a discrepancy between the apparent
function of Bowl and its site of expression: Bowl is necessary to
inhibit/lower Bab2 expression in t1/t2 and t5, but is not present in these
regions in late-third-instar/early-pupal discs. Nevertheless, the effects of
bowl mutations on Bab2 and Dac are strictly cell autonomous
(Fig. 4B,D-F). These
observations can be reconciled if Bowl (and likewise Odd) is expressed within
the cells that give rise to t1/t2 and t5 at early stages when the domains of
the regional genes (bab, dac and Bar) are first established.
This expression must subsequently disappear from these regions and become
restricted to the boundaries of the tarsus.

Early bowl/odd expression

If Bowl and Odd are regulating tarsomere segmentation via effects on
regional genes like bab2, they should be expressed at the boundaries
of the Bab2 domain during early stages. At ∼76-80 hours, both Bowl and Odd
are first detected in a 2-3-cell-wide ring that surrounds the Bab2 expressing
cells and corresponds to the proximal edge of the Dll domain
(Fig. 5A-A″) and the
distal edge of the Dac domain (data not shown). Most of these Bab2-expressing
cells also express BarH1; on the proximal side only a 1-2-cell-wide ring
contains Bab2 and not BarH1 (Fig.
6D). At this stage, therefore, the tarsus consists primarily of
one identity, which has Bab2 and BarH1 expression and appears to approximate
to t4. This early domain is surrounded by cells expressing Bowl and Odd.

Subsequently, a further ring of Bowl and Odd-expressing cells appears in
the centre of the Bab2 domain, at the boundary with the pretarsus, and Bab2 is
rapidly lost from within this ring (Fig.
5B-B″). Bab2 is now flanked both proximally and distally by
Bowl/Odd. At later stages, gaps appear between Bab2 and the flanking rings of
Bowl/Odd (Fig. 5C-C″).
These gaps expand and, at the same time, Bab2 expression becomes more graded,
with decreasing levels at the edges of its expression domain. This is most
marked in the proximal (t2) direction, and is even more evident with Bab1 than
with Bab2 (Couderc et al.,
2002). Dac expression also extends distally beyond the proximal
ring of Bowl/Odd (data not shown) so that it occupies t1 and a small part of
t2 by the time the leg disc everts. As a consequence, a series of distinct
territories is established within the tarsus by late third instar. Bowl/Odd
mark the extreme (tibia/t1 and t5/pretarsal) boundaries of the tarsus and Bab2
expression spans t2-t5 with the peak of its expression in t3/t4.

Both the expression patterns and the phenotypes suggest that cells within
the t1/t2/t3 and t5 regions of the tarsal domain contain Bowl/Odd at 76-86
hours. We propose that Bowl/Odd expression is gradually lost from the tarsal
cells as they proliferate, giving rise to a temporal gradient of Bowl/Odd
(prolonged expression in t1, shorter period of expression in t3/t2). If this
is the case, expression from the odd-lacZ line might be visible
within t1/t2/t3 because of the endurance of β-galactosidase. Indeed, in
96-hour-old odd-lacZ discs, we detect β-galactosidase at lower
levels within many cells of the tarsus
(Fig. 5G). We cannot
definitively show a temporal gradient by this method, but the expression of
odd-lacZ is most persistent close to the final domain of Bowl and
Odd, consistent with this model.

Ectopic bowl causes expansion of proximal and distal tarsal
fates

Because bowl mutations result in expansion of `central tarsal'
(t3/t4) fates, we anticipated that persistent expression of Bowl within the
tarsus would have the converse effect, expanding proximal (t1/t2) and distal
(t5) fates. To test this we used GAL4 driver lines to direct expression of
UAS-bowl within the tarsus, scoring phenotypes in the adult male
pro-thoracic legs, using the sex comb as a marker of t1
(Fig. 5A-D). Expression of
UAS-bowl throughout the tarsal region (Dll-Gal4) gave rise to legs
with expanded t1 fates manifest in the ectopic sex-combs on distal tarsal
segments. In the more strongly expressing lines, the tarsus became severely
distorted and carried sex-comb bristles throughout its length
(Fig. 6C,D). Even with more
restricted production of Bowl (e.g. klumpfuss-GAL4)
(Klein and Campos-Ortega,
1997), similar transformations occurred, with ectopic sex-comb
bristles present in t2 and t3 (Fig.
6B).

To determine whether expansion of t1 fates occurs at the expense of
`central tarsal' fates, we assayed the effects of ectopic Bowl on Bab, Dac and
BarH1. In leg discs from Dll-GAL4/UAS-bowl, levels of Bab2 were
strongly reduced and more patchy than in the wild type, consistent with
central tarsal identity being compromised
(Fig. 6E,F′). Conversely,
the domains of Dac and BarH1 were extended so that they were almost contiguous
in the middle of the tarsus (Fig.
6F,F′), demonstrating the expansion of t1 and t5 fates.
Ectopic expression of Bowl in a more restricted domain (e.g. with
Ptc-Gal4) also reduced Bab (Fig.
5G-G″) specifically within the domain of ectopic expression.
The inhibition of bab2 by Bowl fits with the phenotypes of
bab/bab2 loss-of-function alleles, which are similar to that of
ectopic Bowl (ectopic sex combs on distal segments)
(Godt et al., 1993;
Couderc et al., 2002). In
analysing the levels of Bowl produced by the directed misexpression, we noted
that high levels of protein only accumulated close to the normal sites of
expression. Elsewhere, such as within the tarsus, protein levels remain low
and patchy (Fig. 6G′),
even though mRNA levels are fairly uniform throughout the domain of
misexpression (data not shown). Despite the low levels of protein, we still
see inhibition of Bab within the tarsus
(Fig. 6G″).

Further support for the role of bowl in patterning the proximal tarsus
comes from analysing spineless mutant legs. This gene is essential
for antenna development but is also expressed transiently in the tarsus of the
early third-instar leg (Duncan et al.,
1998). The phenotype observed in weak spineless mutants
(ssa/ss114;
Fig. 5H,H′) resembles
that of ectopic Bowl (with ectopic sex combs in t2 and an ectopic joint within
t1), and we find that the domain of Bowl expression remains broader in
spineless larval discs and that ectopic patches of Bowl persist in t1
and t2 of early pupal discs (Fig.
6I,I′). Sometimes, the ectopic Bowl forms a discrete ring
within t1 that corresponds to the site of the ectopic joint. Persistent Bowl
therefore alters P/D patterning, promoting t1-like fates and, in some cases,
resulting in an ectopic tibia/t1-like joint. These data suggest that
spineless is involved in keeping Bowl absent from in the tarsus. In
agreement with this, ectopic Spineless results in loss of Bowl (data not
shown), although these conditions also result in transformation to antenna
fates, complicating the interpretations.

Discussion

P/D patterning in the Drosophila limb involves the subdivision of
the primordium into concentric regions through the expression of `gap' genes,
whose expression is initiated in response to Dpp and Wg
(Lecuit and Cohen, 1997).
Subsequent pattern builds on this initial subdivision. Here, we have shown
that the genes bowl and odd are involved in a novel aspect
of this process that elaborates the pattern within the tarsus to generate the
correct number and structural diversity of the tarsomeres. Mutations in
bowl or odd cause cells at the proximal and distal positions
in the tarsal region to acquire fates of more centrally placed cells, giving
rise to truncated or fused tarsomeres. Conversely, ectopic Bowl leads to a
transformation of central fates to more proximal or distal fates, again
causing distortions and truncations of the tarsus. The changes in fate are
manifest in the expression patterns of genes such as bab1 and
bab2, which are normally present at the highest levels in t3/t4
tarsomeres and at lower levels in t2 and t5
(Godt et al., 1993;
Couderc et al., 2002). Absence
of bowl leads to elevated Bab2 levels in t2 or t5 and to expression
in proximal regions (t1), where bab2 is normally silent. One notable
feature of Bab1/Bab2 expression is that it is modulated into rings of higher
and lower expression (Godt et al.,
1993). This modulation is also partially lost in bowl
mutant clones (and in Dac mutants)
(Chu et al., 2002), arguing
that it is intimately associated with the elaboration of patterning.

Previous studies have shown that bab1/bab2 expression is promoted
by Dll and that its proximal and distal limits are dependent on Dac proximally
and on epidermal-growth-factor-receptor signalling distally
(Campbell and Tomlinson, 1998;
Campbell, 2002;
Couderc et al., 2002;
Galindo et al., 2002). We
propose that these activities not only define the initial domain of
bab1/bab2 expression but also indirectly regulate the
production of Bowl and Odd through their effects on Notch-ligand expression
(Rauskolb, 2001). Bowl is then
necessary to fine tune bab2 expression so that its levels are low or
absent in the extremities of the tarsus, allowing these to adopt t1 and t5
characteristics (Fig. 7). If
one of the factors responsible for positively regulating bab1/bab2
expression was present transiently, its decay would also contribute to the
gradation in Bab2 expression and could explain why Bab2 is not turned on in
the t1 cells that have lost Bowl at late stages.

Model of the relationship between Bowl and Bab2 expression domains and limb
patterning. (1) Early stage of tarsal development: a leg imaginal disc with
tarsal domains of Bab2 (blue) and Bowl (green). Dotted line outlines region
shown below and in subsequent stages. Within this region (rectangle), Bab2
expression (blue) is uniform and flanked by Bowl (green). Bowl inhibits Bab2.
Bab2 (or another target of Bowl) prevents upregulation of Dac and BarH1
(dashed grey lines). Below is shown the hypothetical distal leg structures
correlating with this stage of expression (Ti, tibia; ta, tarsus; pt,
pretarsus), the tarsus consists of a single segment. Arthropods with an
unsegmented tarsus are predicted to arrest distal limb patterning at this
stage. (2) Middle stage of tarsal development. The length of the tarsal
territory (rectangle) has increased. Bab2 expression (blue) is now induced in
a larger domain as Bowl (green) decays from the central region (top).
Hypothetical distal leg structures correlating with this stage of expression,
the tarsus consists of three segments. Arthropods with intermediate numbers of
tarsal segments are predicted to arrest distal limb patterning at this stage
(bottom). (3) Late stage of tarsal patterning. The length of the tarsal
territory (rectangle) has further increased. Bab2 expression (blue) has
reached its full extent; as Bowl (green) decays further, Bab2 can no longer be
induced and Dac and BarH1 are upregulated in t1 and t5, respectively (top).
Distal leg structures correlating with this stage of expression; the tarsus
consists of five segments (bottom).

The effects of Bowl and Odd on tarsal development were initially difficult
to reconcile with their expression. In late stages of limb development (late
L3/early pupal), the proteins are only present at sites of Notch activity
outside the tarsus, not within the tarsus, even though the most obvious
phenotypes are tarsomere fusions. All of the sites of expression are
precursors for the `true' joints (those with tendon attachments and direct
muscle control) (Snodgrass,
1935), suggesting that Bowl/Odd could have a primary role in the
establishment of joints and that the regulation of tarsal patterning has been
acquired secondarily. We propose that effects on patterning occur because the
proximal and distal parts of the tarsus are formed by cells that synthesize
Bowl/Odd at an earlier stage and that the levels of Bowl/Odd determine the
extent of tarsal gene expression (Fig.
7). When the tarsus is first defined by the expression of
bab, Bowl/Odd directly flank this domain. As the tarsus expands, Bowl
and Odd are only retained at the boundary and are lost from the intervening
cells; as a consequence, bab2 is derepressed. In this way, cells
closest to the initial domain of Bab2 expression would contain Bowl/Odd for
the least time and therefore have higher levels of Bab2 than those closer to
the tibial boundary. A similar relationship between expression and phenotype
has been seen with drumstick (drm; a gene related to
bowl and odd that is required for hindgut morphogenesis)
(Green et al., 2002). At late
embryonic stages, drm expression is only detected in the most
anterior cells of the small intestine, even though it influences cell
behaviour along the whole length of the intestine. By tracing earlier phases
of expression, Green et al. were able to show that drm is transiently
expressed more broadly and gradually becomes restricted to the anterior
hindgut boundary (Green et al.,
2002), which is similar to what we observed for odd-lacZ
expression in the leg. It is possible that these similarities in drm,
odd and bowl regulation reflect a common underlying mechanism
conserved between hindgut and leg morphogenesis.

Notch activation appears to be one key factor in promoting the accumulation
of Bowl and Odd at the tarsal boundaries
(Fig. 3) (see
Rauskolb, 2001), but some data
indicate that other factors are required and that the regulation might be
indirect. First, Bowl and Odd can only be induced at a subset of the locations
where Notch is active, so Notch alone is not sufficient. Second, although all
Notch clones at the t5/tibia boundary result in a loss of Bowl protein, not
all clones at the more proximal boundaries have a phenotype. Because the
smaller clones tend to have the least effect on Bowl, Notch appears to
initiate but not to maintain Bowl expression at these locations. Third,
although regulation of odd can be fully explained by its effects on
transcription, Bowl might be subject to post-transcriptional regulation. When
we drive expression of bowl mRNA through the leg (using GAL4
drivers), we detect at best low levels of Bowl protein within the tarsus,
suggesting that the translation or the stability of the protein are regulated.
Candidates to participate in Odd and Bowl regulation include Spineless
(Fig. 6) and Lines, a protein
that acts antagonistically to Bowl and Drm in hindgut morphogenesis
(Iwaki et al., 2001;
Green et al., 2002). Although
the combined actions of Notch and these factors might explain the initial
expression of Bowl and Odd, the mechanism that maintains their expression
specifically at the boundaries of the tarsus is unclear. This aspect of
regulation is crucial for the diversification of the tarsomeres and, if our
model is correct, would be linked to proliferation. Our predictions are that
tarsal cells should show a bias in their patterns of proliferation, as is the
case in more proximal regions of the leg
(Weigmann and Cohen, 1999),
and that the progeny of Bowl-expressing cells should occupy the t1/t2 and t5
tarsal segments. We have not yet been able specifically to monitor the
proliferation pattern and fate of Bowl-expressing cells to test these
predictions.

One extrapolation from our proposed model for tarsal development in
Drosophila is that the basal or ancestral state consisted of a single
tarsal segment, specified by uniform levels of Bab and directly flanked by
sites of Bowl expression prefiguring the tarsal/tibial and tarsal/pretarsal
joints. This is in agreement with the phylogenetic evidence, which points
towards the ancestral arthropod limb having an unsegmented tarsus (as remains
the case for many modern arthropods, including some insects)
(Snodgrass, 1935).
Furthermore, there is considerable variation in the extent of tarsal
subdivision, with most insects having between two and five tarsomeres (some
arachnids have further subdivisions;
Snodgrass, 1935). These
differences in pattern could be explained by differences in either the
duration or the rate of proliferation during the crucial phase when
bowl/odd influence bab2 patterning. Although mutations in
Notch or bowl/odd affect the extent of tarsal proliferation,
as do mutations in spineless and bab2, none of these
activities alone is sufficient to cause an increased length of the tarsus
(although ectopic Notch activity can give ectopic outgrowth)
(Rauskolb and Irvine, 1999).
Further investigation of how these factors combine to coordinate tarsal
patterning and proliferation should help us to unravel the mechanism
underlying the diversification of arthropod limb structure. Furthermore, as
modifications of bab2 expression are correlated with diversification
of pigmentation and trichome patterns in Drosophila species
(Gompel and Carroll, 2003),
the possibility that bab2 expression is intrinsic to diversification
of tarsal patterning suggests that changes in the regulation of a single gene
could contribute to the diversification of many different morphological
traits.

Acknowledgments

We thank J. de Celis, D. Strutt and I. Duncan for providing fly stocks, E.
Knust, F. Laski and K. Saigo for generously supplying us with antibodies, E.
Harrison for help with fly injections, and M. Furriols and other members of
our laboratory for much helpful advice and discussions. We are also very
grateful to N. Brown, S. Cohen and C. Baker for their comments on the
manuscript, and to C. Rauskolb and V. Hatini for valuable discussions and
sharing of data prior to publication. This research was supported by a project
grant from the Medical Research Council.

Footnotes

Note added in proof

A related paper by Hao et al. discussing the expression and function of
bowl-related genes in the Drosophila leg is currently in press
(Hao et al., 2003).

Godt, D., Couderc, J. L., Cramton, S. E. and Laski, F. A.
(1993). Pattern formation in the limbs of Drosophila:
bric-a-brac is expressed in both a gradient and a wave-like pattern
and is required for specification and proper segmentation of the tarsus.
Development119,799
-812.

Klein, T. and Campos-Ortega, J. A. (1997).
klumpfuss, a Drosophila gene encoding a member of the EGR
family of transcription factors, is involved in bristle and leg development.
Development124,3123
-3134.

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